Denatured macroprotein with divalent tin for tagging with technetium-99m and method of preparation

ABSTRACT

An improved composition suitable for tagging with technetium-99m and use in lung scanning procedures consists essentially of an injectable suspension in buffer solution of particles of a denatured macroprotein having divalent tin bound thereto. The macroprotein has a molecular weight of at least about 20,000. A method for preparing the injectable suspension by treating macroaggregates or microspheres of a denatured macroprotein with a solution containing divalent tin ions is also provided.

United States Patent 1 [1 1 Wolfangel I DENATURED MACROIROTEIN WITHDIVALENT TIN FOR TAGGING WITH TECHNETIUM-99M AND METHOD OF PREPARATION[75] Inventor: Robert G. Wolfangel, St. Louis. Mo.

[73] Assignee: Mallinckrodt Chemical Works. St.

Louis. Mo.

[22] Filed: Mar. 20, 1972 [2]] App]. No; 236.159

OTHER PUBLICATIONS Lin et al., Journal of Nuclear Medicine, Vol. 12. No.5 (1971) pp. 204-21]. Eckelman et al.. Journal of Nuclear Medicine, Vol.

[ Jan. 28, 1975 12. No. 11 (197]) pp. 707-710.

Morcellet. Use of TinTc 99rn-Nuclear Sci. Abs. Vol 24. No 4.. p. 617.Feb. 28. 1970. Item No. 6078. Charamza. Chemical Abstracts. Vol. 72.1970 p.86, Item No. 87066.

De Paoli. Nuclear Sci. Abs.. Vol. 20. No. 23 Dec. 15. 1966. Item No,43390 Albumin Macroaggregatcs etc."

Primary Examiner-Benjamin R. Padgett Attorney, Agent. or FirmKocnig.Senniger, Powers. and Leavitt ABSTRACT An improved composition suitablefor tagging with technetium-99m and use in lung scanning proceduresconsists essentially of an injectable suspension in buffer solution ofparticles of a denatured macroprotein having divalent tin bound thereto.The macroprotein has a molecular weight of at least about 20,000. Amethod for preparing the inject-able suspension by treatingmacroaggregates or mierospheres of a denatured macroprotein with asolution containing divalent tin ions is also provided.

14 Claims, No Drawings DENATURED MACROPROTEIN WITH DIVALENT TIN FORTAGGING WITH TECHNETIUM-99M AND METHOD OF PREPARATION BACKGROUND OF THEINVENTION This invention relates to the field of radiopharmaceuticaldiagnostic testing and more particularly to an injectable suspension ofsolid particulate denatured macroprotein suitable for tagging withtechnetium-99m and use in lung scanning procedures.

Macroaggregated macroproteins have found use as carriers forradionuclides useful in diagnostic radioscanning techniques. Inparticular, such proteins, for example human serum albumin labeled ortagged with a radionuclide, have been used in the diagnosis of pulmonarycirculatory defects. The distribution in the lungs of radioactivelylabeled protein macroaggregates, as determined by isotope radioscanning,indicates the presence or absence of certain pathological conditions.

To be useful in lung scanning, radioactive macroprotein particles mustbe labeled with a gamma emitting radionuclide and be within a prescribedsize range. The labeled particles must also be non-toxic andbiodegradable, i.e., physically or chemically removable from the lungsin a relatively short period.

Macroaggregated human serum albumin tagged with iodine-131 has becomewidely used for perfusion lung scanning. While the biological propertiesof this scanning agent are excellent, an important objection to its usearises from the decay scheme of iodine-l 3 I. In the course of decay,iodine-131 emits a beta particle, its gamma rays are difficult tocollimate effectively, and it has an 8-day physical half-life. As aresult of these characteristics, a patient receiving an injection ofiodine- 13l-tagged albumin is exposed to a high radiation dose, not onlyto the lungs but to other organs such as the thyroid gland as well.

A radionuclide which may advantageously be substituted for iodine-131 inlung scanning is technetium- 99m which emits only gamma rays which canbe easily collimated, and has a physical half-life of only 6 hours.Chiefly on the basis of favorable dosimetry profiles, a host oftechnetium-99m labeled particles have been prepared and used in lungscanning. A comprehensive review of the present state of knowledge inthis field was published by G. V. Taplin and N. S. MacDonald in Seminarsin Nuclear Medicine, Vol. I, No. 2 (April), 1971, pages 132-52.

The safety, convenience and effectiveness of the lung scanningtechniques employing technetium-99m have heretofore been limited,however, by problems associated with the preparation oftechnetium-tagged albumin. To prepare serum albumin for use in lungscanning, it must be both denatured and precipitated in the form ofmacroaggregates or microspheres of a particular range of particle size.To prepare the albumin in such form, a solution of albumin is typicallyheated to denature the protein after which the solution is cooled andthe albumin precipitated by adjusting the pH to the isoelectric point(approximately pH 4.9). Particles of the desired size are obtained bysuitable control of both pH and temperature. Alternatively, an aqueoussolution of albumin can be dispersed in hot, waterimmiscible oil toprecipitate the albumin in the form of microspheres. Of the severalknown methods for tagging protein particles with technetium-99m, themost common has involved first tagging a solution of albumin withtechnetium and then precipitating to form the solid albumin in particlesofthe desired size. Where this method is utilized, however, an immediatemicroscopic examination of the particles must be made to assure theirproper size, and purification is required to remove unreacted technetiumfrom the suspension. Moreover. because of the short half-life oftechnetium-99m, sterility tests cannot be completed on the denatured andprecipitated protein before it is used in human patients.

In an alternative method for preparing technetium- 99m taggedmacroaggregates, a technetium sulfur colloid is formed in the presenceof denatured albumin microspheres causing the spheres to become taggedwith the technetium. However, labeling is not very efficient by thismethod, and several washes of the protein particles are required toremove the unbound technetium. Also, the tagged microspheres produced bythis method are prone to stick together and it becomes necessary todisperse the resulting agglomerates by an appropriate technique such asultrasonic treatment, immediately prior to injection into a patient.Furthermore, the bond between the technetium and the microspheres issomewhat labile, and if the suspension is allowed to stand unused for aslittle as an hour or so after preparation, both the purification andultrasonic treatment must be repeated before injection.

As a consequence of the various disadvantages outlined above,technetium-tagged albumin has not been found generally useful as a lungscanning agent, despite its desirable radiation properties.

SUMMARY OF THE INVENTION Among the objects of the present invention maybe noted the provision of an improved composition which may be readilyand efficiently tagged with technetium- 99m to produce an injectablesuspension for use in lung scanning procedures; the provision of such acomposition which can be stored for substantial periods of time withoutdegradation, be tagged with technetium-99m and then used for injectionwithout particle size examination, ultrasonic treatment or purification;and the provision of processes for preparing the composition. Otherobjects and features will be in part apparent and in part pointed outhereinafter.

In essence, therefore, the present invention is directed to acomposition suitable for tagging with technetium-99m and use in lungscanning procedures. The

'composition consists essentially of an injectable suspension in abuffer solution of particles of a denatured macroprotein having divalenttin bound thereto, the macroprotein having a molecular weight of atleast about 20,000. The invention is further directed to a process forpreparing the composition. In this process, particles of a denaturedmacroprotein are contacted with a solution containing a water-solublesalt of divalent tin causing divalent tin to be bound to themacroprotein. The macroprotein having divalent tin bound thereto is thenwashed and suspended in buffer solution.

DESCRIPTION OF THE PREFERRED EMBODIMENTS It has now been discovered thatan injectable suspension ofa denatured and precipitated macroproteinhaving divalent tin bound thereto may be prepared and stored withoutdegradation for substantial periods prior to use. Even moresignificantly, it has been found that the macroprotein of the suspensionmay be quickly and easily tagged with technetium-99m and that theresulting suspension of the tagged protein may be safely and effectivelyused in lung scanning techniques without particle size determination.ultrasonic treatment or purification for the removal of unboundtechnetium. Thus, the relatively cumbersome and time-consumingtechniques previously required are avoided and both the cost ofpreparing a suitable suspension for use in lung scanning procedures andthe risks associated with its use are minimized.

The macroprotein constituent of the composition of the invention ispreferably denatured human serum albumin, a protein which isparticularly suitable for use in lung scanning. It will be understood,however, that any denatured macroprotein having a molecular weight of atleast about 20,000 may have divalent tin bound thereto in accordancewith the invention to render it more susceptible to labeling with aradioactive isotope of technetium.

The particles of the protein constituent of the composition of theinvention are characterized as macroaggregates. Macroaggregatcs areconsidered to be particles of irregular form having an average diameterof between about 5 and about 80 microns. Where the protein particles areintended for use as lung scanning agents, the mean size of the particlesis preferably between about l5 and about 30 microns.

The improved susceptibility of the suspended protein macroaggregates totagging with technetium99m relates to the divalent tin which is bound tothe protein. When a suspension of serum albumin treated with divalenttin in accordance with the invention is subsequently treated with asolution containing heptavalent technetium-99m, for example a solutionof sodium pertechnetate, upwards of 90% of the technetium is rapidly andfirmly bound to the serum protein.

In the practice of the invention, human serum albumin or otherappropriate macroprotein is first denatured and then precipitated toform particles of the desired size. Various methods are known to the artfor producing macroaggregates 'or microspheres of denatured protein.While any of these methods may be successfully used, excellent resultshave been obtained using a method wherein a dilute aqueous solution ofhuman serum albumin is first heated at a temperature of about '70l00C.,preferably about 85C., to denature the protein, then cooled to atemperature on the order of l8-22C., preferably about 20C. The albuminis then precipitated by slowly adding a dilute acid, approximately 0.1N,until the pH is about 4.8-5.2, i.e., the isoelectric pH of the proteinbeing about 4.9. Hydrochloric acid is preferred for use in adjusting thepH to precipitate the denatured protein. However, other mineral acids oracetic acid may also be employed. The particle size of themacroaggregates thus precipitated is typically in the range of 5-80microns with a mean particle size of -30 microns. The integrity of theprecipitated aggregates is further improved if the suspension isreheated for a short period of time following precipitation. Preferablythe reheating temperature is of approximately the same order as thedenaturing temperature.

After reheating, the macroaggregated albumin particles are separatedfrom the suspension, washed and resuspended in a fresh buffer solutionhaving essentially the same pH as the isoelectric pH of the protein(i.e.,

pH 4.9). A convenient buffer solution is an acetate buffer having a pHof 4.9 (0.052N sodium acetate and 0.02N hydrochloric acid).

Binding of the protein aggregates with divalent tin is effected byadding to the protein suspension an aqueous solution containing stannousions. Contact of the macroaggregates with stannous ions causes divalenttin to become bound to the protein. For this purpose, stannous chlorideis preferred because of its solubility and greater stability, althoughother watersoluble stannous salts, such as stannous sulfate. may also beused. lt is necessary. however. to employ a divalent tin solution sincetetravalent tin ions are not effective.

Sufficient stannous ions are available for binding to the protein if themixture of the protein suspension and stannous salt solution containsapproximately 3 mg. of stannous ions per mg. of macroaggregates. Withsuch proportions, binding of divalent tin to the protein molecule occursquickly at room temperature and appears to be substantially complete inabout 15 minutes or so. For optimum results, however, the mixturecontaining stannous ions and protein should be allowed to stand for alonger time, preferably about 24 hours. After the treatment period iscomplete, the aggregates are separated from the liquid phase, washedwith and rcsuspended in fresh buffer solution. After resuspcnsion, themacroaggregates may be stored for a substantial period at 4C.

In an alternative embodiment of the invention, the precipitation andtin-binding steps of the process are combined into a single step.Following this alternative, the stannous salt is dissolved in the diluteacid used to precipitate the heat-denatured protein thus eliminating onestep of the above-described process. Whichever procedure is followed,the same end result is realized, i.e., the preparation of a suspensionof precipitated de natured protein particles having divalent tin boundthereto.

The resulting suspensions have a shelf life of approximately 60 dayswhen stored at 4C. before such characteristics as particle integrity andsusceptability to radionuclide tagging begin to deteriorate. If 60 daysof storage are significantly exceeded, the tagging efficiency normallyfalls off to the extent that the product should be discarded.

The shelf life of the macroaggregate suspensions may be extended beyond60 days by freezing the suspension and storing it in the frozen state.The shelf life of the frozen suspension has been found to extend toapproximately 4 months or longer. Storage in the frozen state has notgenerally been attractive, however, since the macroaggregate particlestend to clump together during subsequent thawing and form agglomerateswhich are too large for use in lung scanning procedures.

This problem is overcome by incorporating into the suspension betweenabout 8% and about 16% by weight of an antiagglomerating agent such aspropylene glycol, glycerin, gelatin or sucrose. The preferredantiagglomerating agent is propylene glycol. Agglomeration of themacroprotein particles is effectively prevented by inclusion of theanti-agglomerating agent in the suspension immediately prior tofreezing.

To prevent bacterial growth in the suspension, the addition of asuitable preservative is also advisable. For this purpose, the additionof approximately 1% by weight of benzyl alcohol to the initial proteinsolutions or to the finished suspensions of the invention has been foundacceptable and effective. Other nontoxic bacte riostatic preservativesmay be substituted for benzyl alcohol.

The tin-treated protein of the suspension is conveniently and easilytagged with technetium-99m by adding the sodium pertechnetate eluatefrom a technetium generator directly to the suspension. Generatorssuitable for preparation of the eluate are commercially available. Forexample, one such generator is described in Shumate US. Pat. No.3,535,085, dated October 20, 1970. When the eluate and suspension aremixed, binding of the technetium to the protein occurs spontaneously andquickly with an efficiency of 95% or more. Preferably, about 30 minutesare allowed to optimize the specific activity of the tagged suspension.The mechanism of tagging is not clear but it involves oxidation of thedivalent tin to the tetravalent state with concomitant reduction of theheptavalent technetium to a lower valency. Tetravalent tin remainsattached to the protein. Within minutes of the addition of thetechnetium eluate thereto, the suspension of tagged albumin may beintravenously injected directly into animals or humans without anyfurther treatment.

The effectiveness of lung scanning is optimized by using sufficienteluate to provide a tagged suspension having a specific activity of upto approximately 20 mci TcO /mg. of the macroaggregated albumin. Fiveminutes after injection, the lungs of a subject contain 90% or more ofthe injected technetium-99m. The presence or absence of a pathologicalcondition is then determined by radioscanning the lungs of the patientand comparing the emission pattern thereof with a standard pattern.

The tagged denatured albumin is eliminated from the subjects lungs at arate corresponding to a biological half life of approximately 510 hours.

Thus, the suspensions of the tin-treated albumin macroaggregates may bestored for substantial periods of time while still retaining theirpotency. Then, when desired, they may be tagged with technetium merelyby adding a solution containing sodium pertechnetate for immediate useas a lung scanning agent without further treatment. The invention thusobviates the costly, timeconsuming and difficult procedures which haveheretofore been necessary.

The following examples illustrate the invention.

EXAMPLE 1 A solution containing fresh human serum albumin (500 mg.) in a1% by weight aqueous solution of benzyl alcohol (45 cc.) was heated forminutes at 85C. with slow stirring to denature the protein. The solutionwas then transferred to a cold water bath, and when the temperature hadreached l8-22C. 2 ml. of 0.085N hydrochloric acid solution was slowlyadded over a period of about 3 minutes with rapid stirring. The pH ofthe resulting suspension of precipitated albumin was 4.8 to 5.2. Thesuspension was then reheated to 83C. for 5 minutes with slow stirring,causing the precipitated albumin aggregates to become more compact andless subject to fragmentation. The particle size analysis of theprecipitated macroaggregates indicated the size range to be 5-80microns, with a mean of 15-30 microns. After reheating, themacroaggregates were collected by centrifugation and resuspcndcd in 30ml. of an acetate buffer solution having a pH of 4.8 (0.052N sodiumacetate and 0.02N hydrochloric acid).

EXAMPLE 2 To the suspension produced in Example 1 was added 0.6 ml. ofa1N HCl solution containing 50 mg. of stannous chloride per ml. This isequivalent to approximately 3 mg. of divalent tin per I00 mg. ofalbumin. The pH of the resulting mixture was 4.04.l. Two ml. of a 0.4Msodium acetate solution was added, and the mixture allowed to incubatefor 24 hours. After the incubation period was complete, the proteinmacroaggregates were washed three times by centrifugation, withresuspension in 30 ml. of fresh acetate buffer following each washing.The treated particles were finally resuspended in 50 ml. of pH 4.8acetate buffer containing 1% by weight benzyl alcohol.

The resulting suspensions may be stored at 4C. or frozen as previouslydescribed. If they are to be stored at 4C., the suspensions are diluted1:5 with acetate buffer (pH 4.8) containing 1% benzyl alcohol. Iftheyare to be frozen the suspensions are diluted I15 with acetate buffer(pH 4.8) containing 1% benzyl alcohol and 8-16% propylene glycol, forexample.

ln view of the above, it will be seen that the several objects of theinvention are achieved and other advantageous results attained.

As various changes could be made in the above methods and productswithout departing from the scope of the invention, it is intended thatall matter contained in the above description shall be interpreted asillustrative and not in a limiting sense.

What is claimed is:

1. A composition suitable for rapid tagging with technetium-99m and usein lung scanning procedures consisting essentially of an injectablesuspension in a buffer solution of particles of denatured macroproteinhaving divalent tin bound thereto, said macroprotein having a molecularweight of at least about 20,000.

2. A composition as set forth in claim 1 wherein the pH of said buffersolution is approximately the isoelec tric pH of said denaturedmacroprotein.

3. A composition as set forth in claim 1 wherein said macroproteincomprises particles of macroaggregated human serum albumin.

4. A composition as set forth in claim 1 wherein the size of theparticles is substantially between about 5 and about microns.

5. A composition as set forth in claim 4 wherein the mean size of saidparticles is between about 15 and about 30 microns.

6. A composition as set forth in claim 1 including macroproteinparticles in the form of microspheres.

7. A composition as set forth in claim 1 wherein the buffer solutioncontains between about 8 and about l6 by weight of an antiagglomeratingagent selected from the group consisting of propylene glycol, sucrose,glycerin and gelatin.

8. A process for preparing a buffered injectable sus pension ofparticles of denatured macroprotein suitable for rapid tagging withtechnetium-99m and use in lung scanning procedures comprising the stepsof:

contacting particles of a denatured macroprotein with a solutioncontaining a water-soluble salt of divalent tin causing divalent tin tobe bound to said macroprotein;

washing the macroprotein having divalent tin bound thereto with a buffersolution; and

12. A process as set forth in claim 8 wherein said macroprotein is inthe form of macroaggregates.

13. A process as set forth in claim 8 wherein said macroprotein is inthe form of microspheres.

14. A process as set forth in claim 8 wherein said macroprotein isprepared by precipitating denatured macroprotein in an acid medium at apH approximately the isoelectric pH of said protein.

2. A composition as set forth in claim 1 wherein the pH of said buffersolution is approximately the isoelectric pH of said denaturedmacroprotein.
 3. A composition as set forth in claim 1 wherein saidmacroprotein comprises particles of macroaggregated human serum albumin.4. A composition as set forth in claim 1 wherein the size of theparticles is substantially between about 5 and about 80 microns.
 5. Acomposition as set forth in claim 4 wherein the mean size of saidparticles is between about 15 and about 30 microns.
 6. A composition asset forth in claim 1 including macroprotein particles in the form ofmicrospheres.
 7. A composition as set forth in claim 1 wherein thebuffer solution contains between about 8 % and about 16 % by weight ofan antiagglomerating agent selected from the group consisting ofpropylene glycol, sucrose, glycerin and gelatin.
 8. A process forpreparing a buffered injectable suspension of particles of denaturedmacroprotein suitable for rapid tagging with technetium-99m and use inlung scanning procedures comprising the steps of: contacting particlesof a denatured macroprotein with a solution containing a water-solublesalt of divalent tin causing divalent tin to be bound to saidmacroprotein; washing the macroprotein having divalent tin bound theretowith a buffer solution; and thereafter suspending in a buffer solutionthe macroprotein having divalent tin bound thereto.
 9. A process as setforth in claim 8 wherein said macroprotein is human serum albumin.
 10. Aprocess as set forth in claim 8 wherein said solution containingdivalent tin ions is a solution of stannous chloride.
 11. A process asset forth in claim 10 wherein said solution contains approximately 3 mg.of divalent tin for each 100 mg. of macroprotein.
 12. A process as setforth in claim 8 wherein said macroprotein is in the form ofmacroaggregates.
 13. A process as set forth in claim 8 wherein saidmacroprotein is in the form of microspheres.
 14. A process as set forthin claim 8 wherein said macroprotein is prepared by precipitatingdenatured macroprotein in an acid medium at a pH approximately theisoelectric pH of said protein.